Long‐term daily oral administration of intestinal permeation enhancers is safe and effective in mice

Abstract Although protein drugs are powerful biologic therapeutics, they cannot be delivered orally because their large size and hydrophilicity limit their absorption across the intestinal epithelium. One potential solution is the incorporation of permeation enhancers into oral protein formulations; however, few have advanced clinically due to toxicity concerns surrounding chronic use. To better understand these concerns, we conducted a 30‐day longitudinal study of daily oral permeation enhancer use in mice and resultant effects on intestinal health. Specifically, we investigated three permeation enhancers: sodium caprate (C10), an industry standard, as well as 1‐phenylpiperazine (PPZ) and sodium deoxycholate (SDC). Over 30 days of treatment, all mice gained weight, and none required removal from the study due to poor health. Furthermore, intestinal permeability did not increase following chronic use. We also quantified the gene expression of four tight junction proteins (claudin 2, claudin 3, ZO‐1, and JAM‐A). Significant differences in gene expression between untreated and permeation enhancer‐treated mice were found, but these varied between treatment groups, with most differences resolving after a 1‐week washout period. Immunofluorescence microscopy revealed no observable differences in protein localization or villus architecture between treated and untreated mice. Overall, PPZ and SDC performed comparably to C10, one of the most clinically advanced enhancers, and results suggest that the chronic use of some permeation enhancers may be therapeutically viable from a safety standpoint.

subcutaneous or intravenous injection. 4 There is a strong incentive to enhance the intestinal absorption of these drugs so they can be delivered orally, which will improve patient compliance and quality of life.
The relevance of oral macromolecular delivery is expected to increase as more protein drugs are approved and other biologics such as nucleic acid-based therapeutics are developed.
The most common strategy to facilitate the intestinal absorption of orally delivered macromolecules is the use of permeation enhancers. 5,6 These molecules affect the intestinal epithelium, which is a single layer of columnar epithelial cells, to increase its permeability. Permeation enhancers typically act through one of two mechanisms. Some enhancers increase paracellular permeability, or the transport between cells, through molecular manipulation of the protein complexes that connect the epithelial cells to one another. 5,7 These protein complexes, called tight junctions, maintain the polarity and barrier function of the epithelium. 8,9 Alternatively, some enhancers act transcellularly, meaning that they increase transport through intestinal epithelial cells. This can occur through upregulation of receptor-specific transport processes or by disrupting the lipid membrane of the epithelial cells via fluidization. 7,10 A number of oral biopharmaceutical formulations containing permeation enhancers are in clinical trials, and the first oral peptide formulation including a permeation enhancer was approved by the FDA in 2019. 11 The permeation enhancers that have made clinical progress have often been limited to generally recognized as safe (GRAS) substances and food additives. For example, sodium caprate (C 10 ) is a native component of dairy products and a common food additive. C 10 has been the subject of numerous cell culture, preclinical, and clinical studies, making it one of the most widely studied permeation enhancers, and its safety in humans is well established. [12][13][14][15][16] However, C 10 is an anomaly, and the majority of permeation enhancers have not been examined for their effects following repeat dosing in animals or in humans.
Because only a handful of permeation enhancers have translated into the clinic, there is pessimism surrounding their utility.
The primary concern is that chronic absorption enhancer use will cause toxicity, either due to cumulative epithelial damage or the unwanted passage of toxic substances into systemic circulation.
However, there are few published studies in animals or humans that validate this concern in a repeat dosing scenario. 17 Most reports on permeation enhancers present efficacy and toxicity data from cell culture experiments or from animal experiments that include only a single dose. 16,18,19 As such, it is not understood how enhancer chemistry and mechanism of action affect patient health following chronic dosing, and this knowledge gap hinders the rational design of next-generation permeation enhancers.
To address this knowledge gap, we evaluated the effects of three oral permeation enhancers in mice that were delivered once daily for 30 days. Here, we show that chronic enhancer dosing did not produce negative health outcomes and that the modest changes observed for some endpoints (e.g., gene expression) resolved after a 1-week washout period. Together, these data suggest that chronic dosing of a broader range of permeation enhancers may be a clinically viable strategy for oral macromolecule and protein delivery.

| One day time point permeability study
Mice were fasted for 12 h prior to the start of the experiment in cages with fasting grates and no food or bedding but free access to water. Negative control mice were orally gavaged with 600 mg/kg 4 kDa FITC-dextran (FD4) dissolved in PBS. Treated mice received either 60 mg/kg 1-phenylpiperazine (PPZ), 200 mg/kg sodium deoxycholate (SDC), or 390 mg/kg sodium caprate (C 10 ) dissolved in PBS in addition to 600 mg/kg FD4. All solutions were dosed at 10 μl/g of body weight. Blood samples were taken from the submandibular vein at 0, 0.5, 1, 1.5, and 3 h postgavage. Blood was collected in serum tubes (VWR, Radnor, PA) and centrifuged at 15,000 Âg for 10 min to isolate the serum. 10 μl of serum was diluted 1:10 with PBS in black 96-well plates, and fluorescence was measured on a BioTEK Synergy H1 plate reader at 490 nm excitation and 520 nm emission wavelengths. A calibration curve of FD4 was prepared for each experiment to calculate the concentration of FD4 in the blood. A blank fluorescence value of serum from an untreated mouse (not one of the subjects of this study) was subtracted from each measurement to account for autofluorescence of biological materials at these wavelengths. After 3 h, mice were sacrificed, the small intestine and colon were dissected, and tissue samples were collected for RNA extraction (see section on qRT-PCR).

| Baseline permeability measurement
One week before the study began, baseline intestinal permeability was measured as follows. Mice were fasted for 12 h prior in cages with fasting grates and no food or bedding but free access to water.
Then, mice were orally gavaged with 600 mg/kg FD4. After 3 h, blood was taken from the submandibular vein and mice were returned to standard cages and fasting was ended. FD4 concentrations in the blood were determined as described above.

| Safety study design
The experiment began when mice were 14 weeks old. Mice were weighed daily, and their condition was monitored. Conditions requiring sacrifice for humane considerations were defined as mice losing 20% or more of their body weight. Mice were randomly assigned to treatment groups prior to the start of the study.
On Day 1 of the study, mice were weighed and received 600 mg/ kg FD4 dissolved in PBS with no treatment, 60 mg/kg PPZ, 200 mg/ kg SDC, or 390 mg/kg C 10 by oral gavage. After 3 h, blood was taken from the submandibular vein, and mice were returned to standard cages and fasting was ended. Blood concentrations of FD4 were eval- On all other days , mice were weighed and received PBS, 60 mg/kg PPZ, 200 mg/kg SDC, or 390 mg/kg C 10 via oral gavage. On Day 30, the protocol for treatment and intestinal permeability measurement was carried out as previously done, and then the treatment groups were randomly subdivided into two groups each. One subgroup (chronic exposure group) was sacrificed on Day 30. Blood was collected by cardiac puncture, the small intestine and colon were removed, and sections were collected for RNA extraction and histology.
The other subgroup (washout group) was returned to normal cages for a one-week recovery period during which mice received no treatment. Mice were weighed daily. On Day 37, mice were weighed and orally gavaged with 600 mg/kg FD4 in PBS with no added permeation enhancers. After 3 h, blood was taken to assess FD4 serum concentrations as described above. Then the mice were sacrificed, and blood and tissue samples were taken as described above.

| Fecal scoring
Mouse feces were collected on each of the permeability measurement days and assigned a score from 0 to 3 based on solidity, presence of mucus, and presence of blood as determined using Hemoccult Guaiac Fecal Occult Blood Test slides.

| Histology
After dissection, small intestine and colon samples were immediately put into 4% formaldehyde for 24 h. Then samples were rinsed with PBS, transferred to 30% sucrose, and stored at 4 C. Samples were embedded in Sakura Tissue-Tek Optimal Cutting Temperature Compound (OCT; VWR) and stored at À80 C. Samples were sectioned to 10 μm thickness on a Shandon Cryotome ® (Thermo Fisher) and then stained as follows. 2.9 | RNA extraction and real-time quantitative polymerase chain reaction After dissection, tissue samples from the small intestine and colon were immediately placed into RNAlater (Thermo Fisher) and stored at À20 C until they were processed. Tissue samples were placed in  Table 1.

| Serum zonulin and TNF-α quantification by ELISA
After the mice were sacrificed, blood was collected by cardiac puncture and centrifuged to isolate the serum. The serum concentrations of zonulin and TNF-α were measured using enzyme-linked immunosorbent assay (ELISA) kits purchased from Abcam. Samples were assessed according to the manufacturer's instructions with a 1:1000 dilution factor for zonulin and a 1:100 dilution factor for TNF-α.

| Statistics
For the time point study, group size was n = 6. For the longitudinal experiment, mice were randomly assigned to treatment groups such that the initial group size was n = 12. All data are presented as the mean with error bars representing the standard error of mean (SEM).
Significance was determined by two-tailed, unpaired Student's t-tests performed in GraphPad Prism 8.

| RESULTS
The goal of this study was to determine whether chronic administration of intestinal permeation enhancers would cause toxicity. We chose to examine two enhancers known to increase intestinal permeability through distinct mechanisms of action. One of the chosen permeation enhancers is phenylpiperazine (PPZ), which we and others have found to improve paracellular permeation by tight junction rearrangement using in vitro (Caco-2 monolayers) and ex vivo (rodent intestinal tissue in Ussing chambers) models. [20][21][22] We also used sodium deoxycholate (SDC), which is a bile salt that enhances permeability via membrane fluidization and tight junction rearrangement as determined using in vitro and ex vivo models. 23,24 Then, we compared them to sodium caprate (C 10 ), as its nontoxicity and efficacy are extensively characterized in literature. 14,15 The enhancer concentrations used in this study were chosen based on literature data and our previous work. 25 For PPZ, the oral LD 50 in rats is published as 210 mg/kg, and we decided upon 60 mg/kg, which increases FD4 absorption while staying below potentially harmful doses.
SDC is an endogenous bile salt secreted by the gallbladder to aid the digestion of fat in the intestine. In humans, one study measured fedstate concentrations between 0.74 and 86.14 mM. 26 For this study, we chose a dose of 200 mg/kg (equivalent to $48 mM, assuming dilution in the volume of the intestine). According to the MSDS, the oral LD 50 for SDC in mice is over 1 g/kg, which is fivefold higher than the dose used in this experiment. The concentration for C 10 (390 mg/kg, corresponding to 200 mM) was chosen based on an extensive compilation of in vivo studies reviewed by Maher et al. 15 Our chosen concentration falls in the range of previously used doses and is well below the oral LD50 published for rats, which is 3.7 g/kg. 15 As is common in the oral protein delivery field, we chose to use FD4 as a model macromolecular drug for this study. FITC-dextrans are excellent model drugs for oral delivery research not only because they are available in a wide range of molecular weights and relatively inexpensive compared to true protein drugs, but also because they are nondigestible. 27 This means that they can be dosed orally and assumed to pass through the acidic and denaturing conditions of the stomach without being degraded.

| All enhancers increased the intestinal permeation of 4 kDa FITC-dextran
Before beginning the month-long safety study, a 1-day experiment SDC caused the largest increase in permeation, with FD4 blood concentration peaking at 30 min and remaining elevated compared to the untreated control until 3 h after administration. C 10 also rapidly increased FD4 blood concentration, which reached its maximum by 30 min. This increased permeation caused by C 10 did not persist as long as that caused by SDC. PPZ also enhanced permeation with statistical significance, although to a lesser degree than C 10 and SDC. All permeation enhancers caused statistically significant increases in the AUC compared to the untreated control, producing fold increases of 1.7, 5.0, and 2.3 for PPZ, SDC, and C 10 , respectively. Because the enhancer doses in this experiment increased the absorption of FD4 after oral gavage, we used them for the remainder of the study. While not statistically significant, SDC and C 10 increased FD4 concentrations by the final day of treatment. However, after the week-long washout period, no differences from the pretreatment intestinal permeability were observed. These findings do not support the common concern that repeated use of permeation enhancers may cause longterm increases in intestinal permeability.
F I G U R E 3 Enhancers did not permanently increase intestinal permeability after 4 weeks of daily oral administration. Baseline untreated intestinal permeability was measured 1 week before treatment began (gray squares-Pre). Mice were dosed with (a) PBS, (b) PPZ, (c) SDC, or (d) C 10 every day for 30 days, and the concentration of FD4 in the blood was measured five times throughout the 30-day period and again after a 1-week washout period (gray squares-Post). Over the course of treatment, SDC and C 10 caused slight increases in permeability that were not significant, while the Control and PPZ groups had no difference between the permeability on Days 1 and 30. The observed increases in FD4 permeability for the SDC and C 10 groups were no longer present after the washout period. n = 6-12, error bars represent SEM. No statistical differences were found between any groups, with significance defined as p < 0.05 by unpaired, two-tailed Student's t-tests.

| Chronic permeation enhancer exposure does not negatively affect GI health indicators
In addition to intestinal permeability, we measured several other indicators of GI health in mice following a 1-month exposure to permeation enhancers. Mice were weighed daily, as well as assessed for general behavioral signs of healthy versus stressed states, including barbering, hunching, and fecal abnormalities including mucus in the stool and diarrhea. Regarding weight, mice across all groups gained between 2 and 9 g over the treatment period, with an average weight gain of 4.2 ± 1.5 g (Figure 4a). Only the PPZ group gained significantly less weight (3.4 g) compared to the control group (5.3 g). We believe, however, that the reduced variability in the weights of the PPZ group may confound a conclusion that reduced weight gain was caused by PPZ treatment.
We measured the serum concentrations of the protein zonulin, an increase in which would indicate increased intestinal permeability. 30,31 These concentrations were assessed by ELISA for mice sacrificed on treatment Day 30 and after the washout period (Figure 4b). None of the treatment groups had significantly different zonulin concentrations compared to the control group. To better understand changes induced by SDC treatment, we also measured TNF-α concentrations for the control and SDC groups on treatment Day 30. However, no differences were found (data not shown), indicating that SDC did not promote local inflammation mediated by TNF-α.
We also assessed stool quality, as it worsens when the intestinal epithelium is damaged. 32 Fecal samples were collected on permeation measurement days and visually inspected for solidity and mucus. Further, blood in the stool was identified using Hemoccult testing. These three measurements were aggregated into a quantitative fecal score consecutive days of diarrhea or stool containing mucus or blood, and hunching that did not resolve within a few minutes after handling.
The only observed instances of barbering were resolved by separating a dominant mouse from its cagemates and housing it separately for the rest of the study. During the study, three mice in the C 10 group and one in the SDC group were sacrificed due to procedural complications resulting from the oral gavage, and one mouse in the PPZ group was sacrificed due to complications from a blood draw.

| Repeated dosing of permeation enhancers affects mRNA expression of tight junction proteins
Based on the data presented in Figure 2, one dose of permeation enhancer changed the gene expression of several tight junction proteins; therefore, we examined the effects of chronic exposure to these enhancers. Tissue samples were collected from the small intestines and colons of mice sacrificed either on treatment day 30 or F I G U R E 6 Intestinal architecture and tight junction protein localization were not affected by 4 weeks of treatment with permeation enhancers. (a) Sections of small intestine from mice receiving PBS, PPZ, SDC, or C 10 were stained for nuclei (blue, Hoechst), the barrier-forming claudin 3 (green, AF488), and the tight junction protein ZO-1 (red, AF594). (b) Separate sections were stained for nuclei (blue, Hoechst) and Factin (red, AF594).
after the washout period, and the mRNA expression of claudin 2, claudin 3, ZO-1, and JAM-A were measured by qRT-PCR ( Figure 5).
On treatment day 30, average JAM-A expression in the small intestine decreased for all enhancer-treated groups, with only SDC and C 10 causing statistically significant differences (Figure 5a). However, these decreases in JAM-A expression did not persist after the washout period, indicating that any changes are not permanent

| DISCUSSION
The clinical translation of oral permeation enhancers has been limited by an incomplete understanding of their mechanisms and impact on long-term intestinal health. There is consistent and significant skepticism that safe permeation enhancer use is possible. [35][36][37] The most commonly cited concerns include (1) that increased intestinal permeability will result in the absorption of toxic molecules from the intestinal lumen and (2) that prolonged use will permanently decrease the barrier function of the intestinal epithelium. 35 We were thus motivated to design a study to address some of these common concerns. Specifically, we characterized the short-and long-term effects of daily oral permeation enhancer administration in mice. We included three permeation enhancers with distinct mechanisms of action. To our knowledge, the paracellular enhancer, 1-phenylpiperazine (PPZ), and the transcellular enhancer, sodium deoxycholate (SDC), have not been studied for safety or efficacy in vivo. 23 These enhancers were compared to sodium caprate (C 10 ), which is an approved food additive and widelyresearched permeation enhancer that fluidizes the lipid membrane of the epithelial cells and rearranges tight junctions. 15 One major finding of this study is that the three enhancers caused no permanent increases in intestinal permeability ( Figure 3). Previous studies of repeat oral administration of C 10 in rodent and dog models found similar results as determined by pharmacokinetic profiling. 38 We noted that SDC and PPZ maintained efficacy each week and did not gradually increase permeability, which would have indicated damage to the epithelium. Studies of PPZ and SDC have been limited to the Caco-2 cell culture model and ex vivo studies using tissue from rats. However, although these models lack the repair mechanisms of the in vivo intestine, recovery of intestinal barrier integrity was observed after a single dose of the permeation enhancer at effective concentrations. [20][21][22]24,39 We noted an increase in FD4 serum concentration variability for SDC-treated mice over the course of treatment, with the same mice having the highest FD4 serum concentrations each week. This observation that individuals respond variably to permeation enhancement is consistent with published studies. 40 Additionally, daily handling of animals and their associated stress responses may have contributed to variability.
One long-standing concern in the field of oral delivery is that repeated dosing of permeation enhancers will allow pathogens to cross the intestinal epithelium. This concept of undesired and harmful xenobiotic absorption with the use of permeation enhancers is thoroughly reviewed by McCartney, et al. 35 Here, we use the marker molecule 4 kDa FITC-dextran, which is much smaller than lipopolysaccharide, endotoxins, or bacteria (>100 kDa In contrast to PPZ's paracellular mechanism, SDC functions via membrane fluidization of the epithelial cells, which causes increased transcellular transport. 7,23,45 Aligning with this mechanistic characterization, tight junction gene expression in SDC-treated mice varied less compared to control mice than it did for PPZ-or C 10 -treated mice. The results of this study suggest that repeated use of three different permeation enhancers did not alter intestinal health or barrier function in a substantive way. One limitation of this study, however, is that we examined only one concentration for each enhancer.
Enhancer behavior and mechanism can vary considerably as a function of concentration 20,22,51,52 ; therefore, caution is needed when extending these findings to other permeation enhancer doses. Additionally, this study examined a single dose per day. More research is needed to determine whether these results apply to formulations dosed multiple times a day, insulin being one prominent example.

CONFLICT OF INTEREST
The authors have no conflicts of interest to declare.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.